Independence of HIF1a and androgen signaling pathways in prostate cancer.

BACKGROUND: Therapeutic targeting of the androgen signaling pathway is a mainstay treatment for prostate cancer. Although initially effective, resistance to androgen targeted therapies develops followed by disease progression to castrate-resistant prostate cancer (CRPC). Hypoxia and HIF1a have been implicated in the development of resistance to androgen targeted therapies and progression to CRCP. The interplay between the androgen and hypoxia/HIF1a signaling axes was investigated. METHODS: In vitro stable expression of HIF1a was established in the LNCaP cell line by physiological induction or retroviral transduction. Tumor xenografts with stable expression of HIF1a were established in castrated and non-castrated mouse models. Gene expression analysis identified transcriptional changes in response to androgen treatment, hypoxia and HIF1a. The binding sites of the AR and HIF transcription factors were identified using ChIP-seq. RESULTS: Androgen and HIF1a signaling promoted proliferation in vitro and enhanced tumor growth in vivo. The stable expression of HIF1a in vivo restored tumor growth in the absence of endogenous androgens. Hypoxia reduced AR binding sites whereas HIF binding sites were increased with androgen treatment under hypoxia. Gene expression analysis identified seven genes that were upregulated both by AR and HIF1a, of which six were prognostic. CONCLUSIONS: The oncogenic AR, hypoxia and HIF1a pathways support prostate cancer development through independent signaling pathways and transcriptomic profiles. AR and hypoxia/HIF1a signaling pathways independently promote prostate cancer progression and therapeutic targeting of both pathways simultaneously is warranted.

The androgen receptor fuels prostate cancer by regulating central metabolism and biosynthesis.

The androgen receptor (AR) is a key regulator of prostate growth and the principal drug target for the treatment of prostate cancer. Previous studies have mapped AR targets and identified some candidates which may contribute to cancer progression, but did not characterize AR biology in an integrated manner. In this study, we took an interdisciplinary approach, integrating detailed genomic studies with metabolomic profiling and identify an anabolic transcriptional network involving AR as the core regulator. Restricting flux through anabolic pathways is an attractive approach to deprive tumours of the building blocks needed to sustain tumour growth. Therefore, we searched for targets of the AR that may contribute to these anabolic processes and could be amenable to therapeutic intervention by virtue of differential expression in prostate tumours. This highlighted calcium/calmodulin-dependent protein kinase kinase 2, which we show is overexpressed in prostate cancer and regulates cancer cell growth via its unexpected role as a hormone-dependent modulator of anabolic metabolism. In conclusion, it is possible to progress from transcriptional studies to a promising therapeutic target by taking an unbiased interdisciplinary approach.

Systematic comparison of microarray profiling, real-time PCR, and next-generation sequencing technologies for measuring differential microRNA expression.

RNA abundance and DNA copy number are routinely measured in high-throughput using microarray and next-generation sequencing (NGS) technologies, and the attributes of different platforms have been extensively analyzed. Recently, the application of both microarrays and NGS has expanded to include microRNAs (miRNAs), but the relative performance of these methods has not been rigorously characterized. We analyzed three biological samples across six miRNA microarray platforms and compared their hybridization performance. We examined the utility of these platforms, as well as NGS, for the detection of differentially expressed miRNAs. We then validated the results for 89 miRNAs by real-time RT-PCR and challenged the use of this assay as a "gold standard." Finally, we implemented a novel method to evaluate false-positive and false-negative rates for all methods in the absence of a reference method.

The cost of reducing starting RNA quantity for Illumina BeadArrays: a bead-level dilution experiment.

BACKGROUND: The demands of microarray expression technologies for quantities of RNA place a limit on the questions they can address. As a consequence, the RNA requirements have reduced over time as technologies have improved. In this paper we investigate the costs of reducing the starting quantity of RNA for the Illumina BeadArray platform. This we do via a dilution data set generated from two reference RNA sources that have become the standard for investigations into microarray and sequencing technologies. RESULTS: We find that the starting quantity of RNA has an effect on observed intensities despite the fact that the quantity of cRNA being hybridized remains constant. We see a loss of sensitivity when using lower quantities of RNA, but no great rise in the false positive rate. Even with 10 ng of starting RNA, the positive results are reliable although many differentially expressed genes are missed. We see that there is some scope for combining data from samples that have contributed differing quantities of RNA, but note also that sample sizes should increase to compensate for the loss of signal-to-noise when using low quantities of starting RNA. CONCLUSIONS: The BeadArray platform maintains a low false discovery rate even when small amounts of starting RNA are used. In contrast, the sensitivity of the platform drops off noticeably over the same range. Thus, those conducting experiments should not opt for low quantities of starting RNA without consideration of the costs of doing so. The implications for experimental design, and the integration of data from different starting quantities, are complex.

Salt-inducible kinase 2 regulates mitotic progression and transcription in prostate cancer.

UNLABELLED: Salt-inducible kinase 2 (SIK2) is a multifunctional kinase of the AMPK family that plays a role in CREB1-mediated gene transcription and was recently reported to have therapeutic potential in ovarian cancer. The expression of this kinase was investigated in prostate cancer clinical specimens. Interestingly, auto-antibodies against SIK2 were increased in the plasma of patients with aggressive disease. Examination of SIK2 in prostate cancer cells found that it functions both as a positive regulator of cell-cycle progression and a negative regulator of CREB1 activity. Knockdown of SIK2 inhibited cell growth, delayed cell-cycle progression, induced cell death, and enhanced CREB1 activity. Expression of a kinase-dead mutant of SIK2 also inhibited cell growth, induced cell death, and enhanced CREB1 activity. Treatment with a small-molecule SIK2 inhibitor (ARN-3236), currently in preclinical development, also led to enhanced CREB1 activity in a dose- and time-dependent manner. Because CREB1 is a transcription factor and proto-oncogene, it was posited that the effects of SIK2 on cell proliferation and viability might be mediated by changes in gene expression. To test this, gene expression array profiling was performed and while SIK2 knockdown or overexpression of the kinase-dead mutant affected established CREB1 target genes; the overlap with transcripts regulated by forskolin (FSK), the adenylate cyclase/CREB1 pathway activator, was incomplete. IMPLICATIONS: This study demonstrates that targeting SIK2 genetically or therapeutically will have pleiotropic effects on cell-cycle progression and transcription factor activation, which should be accounted for when characterizing SIK2 inhibitors.